1. Field of the Invention
The present invention relates to a cell observation device and a cell observation method for observing reactions of cells, and more particularly, to a cell observation device and the like suitable for screening in a development process of medicinal products.
2. Description of the Related Art
It is generally practiced to culture a large number of cells by using a known synchronous culture method (see, for example, Reference 1), to uniformalize states (for example, the timing of a mitotic phase of a cell cycle) of the large number of cells and observe reactions of the cells to stimulation such as the introduction of a drug. According to this method, an average reaction of the large number of cells can be observed.
Reference 1: R. Weimer, T. Haaf, J. Kruger, M. Poot, M. Schmid, “Characterization of centromere arrangements and test for random distribution in G0, G1, S, G2, G1, and early S′ phase in human lymphocytes”, Human Genetics 88:673-682 (1992)
However, the aforesaid synchronous culture method needs very complicated works. Further, in order to study the correlation between initial states (for example, states immediately before the stimulation is applied) of a large number of cells and their reactions, a large variety of specimens composed of cells in different initial states have to be prepared by the synchronous culture method, which requires enormous labor and time.
Here, an experiment conducted by the present inventors will be described. In this experiment, the correlation between the initial states of the large number of cells and their reactions was studied by the combination of conventional methods. First, a large variety of specimens are prepared by the synchronous culture method. Specifically, a cell suspension at a logarithmic growth phase is adjusted to a concentration of 5×105 cells/ml, a thymidine solution is added thereto so that the final concentration (different depending on the kind of the cells) becomes 0.5 to 2.5 mM, and the cells are cultured in a CO2 incubator for 16 to 24 hours. This operation causes the large number of cells in the specimen to synchronize in a boundary of a gap phase (G1 phase)/a DNA synthesis phase (S phase) and in the S phase of a cell cycle. Next, cell culture supernatants (substances on the surface) are removed by centrifugal separation and are returned to a culture solution to be cultured for 15 hours. This operation causes the large number of cells in the specimen to enter the boundary of a gap phase (G2 phase)/a mitotic phase (M phase). Further, a thymidine solution is added so that the final concentration becomes 0.5 to 2.5 mM, and the cells are cultured for 16 to 24 hours in the CO2 incubator. This operation causes the large number of cells in the specimen to synchronize in the boundary of the G1 phase/the S phase. Note that, in order to know in which phase of the cell cycle the large number of cells in the specimen are synchronized, a DNA amount of each of the cells may be measured by using a known flow cytometry after the cells are dyed with propidium iodidle (PI). The inventors of the present invention prepared a large variety of the specimens in different cell initial states (here, phases of the cell cycle) in the above-described manner.
Then, an expression amount of ion channels on cell surfaces was measured in each of a large variety of the specimens. The ion channel is small conductance (SK) type 2 of calcium-activated potassium channels. A known Western blot method using anti-SK2 channel antibody was implemented for the measurement. FIG. 4 shows the measurement result. In FIG. 4, the horizontal axis shows time, with the start time of the GI phase of the cell cycle being defined as 0, and the time of each of the S phase and the G2 phase/M phase can be seen. The vertical axis in FIG. 4 shows relative density of an expression amount of the ion channels. It has been found out from this measurement result that the expression amount of the ion channels increases/decreases depending on each phase of the cell cycle. Generally, the expression amount of the ion channels correlates with the reaction of a cell (in this case, potential change of a cell membrane), and therefore, the above measurement result implies that the reaction of the cells changes depending on each phase of the cell cycle. Specifically, in the case of the SK2 channel, reactivity is high in the G1 phase and reactivity lowers in the S phase and the G2 phase/M phase.
As described above, in order to study the correlation (for example, FIG. 4) between the initial states of cells and their reactions by using the conventional method, a large variety of specimens in different cell initial states has to be prepared by the synchronous culture method, which requires enormous labor and time.